This invention is related to xcfx89-cycloalkyl-prostaglandin E2 derivatives. More particularly, this invention is related to:
(1) xcfx89-cycloalkyl-prostaglandin E2 derivatives of the formula (I) 
xe2x80x83wherein all the symbols are the same meaning as hereafter defined, non-toxic salt thereof, prodrug thereof and cyclodextrin clathrate thereof.
Prostaglandin E2 (abbreviated as PGE2 hereafter) has been known as metabolite in the arachidonate cascade. It has been known that PGE2 has cyto-protective activity, uterine contractile activity, a pain-inducing effect, a promoting effect of digestive peristalsis, an awakening effect, a suppressive effect of gastric acid secretion, hypotensive activity and diuretic activity etc.
In a recent study, it was found that PGE2 receptor was divided into some subtype which possess different physiological roles from each other. At present four receptor subtypes are known and they are called as EP1, EP2, EP3 and EP4 (Negishi M. et al, J. Lipid Mediators Cell Signaling, 12, 379-391 (1995)).
The present inventors investigated to find new compounds which bind on each receptor specifically, we found that the compounds of the present invention could bind strongly on EP2 subtype receptor and achieved the present invention.
The compounds of the formula (I) of the present invention possess a binding activity for EP2 subtype receptor strongly. Therefore, they are useful for prevention and/or treatment of immunologic diseases (autoimmune diseases, organ transplantation, etc.), asthma, abnormal bone formation, neuronal cell death, liver damage, abortion, premature birth or retina neuropathy of glaucoma etc.
Among the compounds of the present invention of the formula (I), compounds which bind weakly on receptor subtypes except for EP2 and another arachidonic acid metabolism receptor (thromboxane receptor, PGI2 receptor, etc.) do not exhibit other effects and therefore, it is thought that such compounds will be useful as medical agents which have less side-effects.
On the other hand, many patent applications of PG derivatives are known. The following application is mentioned for example.
In the specification of U.S. Pat. No. 4,132,738, a compound of the formula (A) 
wherein
R1A and R2A is hydrogen atom;
R3A is hydrogen atom, or together with R4A is a methylene chain of 4 carbon atoms such that a cycloalkyl of 6 carbon atoms inclusive is formed, or together
with R4A is a bicycloalkenyl or bicycloalkyl moiety having the formula 
(in which pA is an integer having a value of from 0 to 1 and qA is an integer having a value of from 2 to 3 and wherein the double bond of such bicycloalkenyl is in the qA bridge);
R4A together with R3A forms a cycloalkyl or bicycloalkyl or bicycloalkenyl as defined above, or together with R5A is a methylene chain of 3 carbon atoms such that a cycloalkyl of 4 carbon atoms inclusive is formed;
R5A is hydrogen atom, or together with R4A forms a cycloalkyl as defined above; and
R6A is hydrogen atom or straight-chain alkyl having from 1 to 8 carbon atoms; are disclosed as having an inhibitory activity on prostaglandin like.
The present invention is related to
(1) xcfx89-cycloalkyl-prostaglandin E2 derivatives of the formula (I) 
xe2x80x83wherein
R is carboxy or hydroxymethyl;
R1 is oxo, methylene or halogen atom;
R2 is hydrogen atom, hydroxy or C1-4 alkoxy;
R3 is (i) hydrogen atom, (ii) C1-8 alkyl, (iii) C2-8 alkenyl, (iv) C2-8 alkynyl or (v) C1-8 alkyl, C2-8 alkenyl or C2-8 alkynyl, each substituted by 1-3 substituents, being same or different, selected from (1)-(5);
(1) halogen atom,
(2) C1-4 alkoxy,
(3) C3-7 cycloalkyl,
(4) phenyl, or
(5) phenyl substituted by 1-3 substituents selected from halogen atom, C1-4 alkyl, C1-4 alkoxy, nitro or trifluoromethyl;
n is 0-4;
is single bond or double bond;
is double bond or triple bond;
is single bond, double bond or triple bond;
with the proviso that,
1) when 5-6 position is triple bond, 13-14 position is not triple bond,
2) when 13-14 position is double bond represent E or Z form; non-toxic salt thereof, prodrug thereof and cyclodextrin clathrate thereof,
(2) processes for the preparation thereof, and
(3) pharmaceutical agents containing such a derivative as an active ingredient.
In the present invention, prodrug means
1) for compounds of formula (I) of the present invention, those in which R represent COOR10 (in which R10 is C1-6 alkyl), i.e., the compounds of formula (IA) 
xe2x80x83wherein all symbols are the same meaning as hereinbefore defined
2) for compounds of formula (I) of the present invention, those in which R represent CONR2R13 (in which R12 and R13 each, independently, is hydrogen atom or C1-6 alkyl), i.e., the compounds of formula (IB) 
xe2x80x83wherein all symbols are the same meaning as hereinbefore defined, or
3) for compounds of formula (I) of the present invention, those in which R represent COOR10 (in which R10 is the same meaning as hereinbefore defined), R1 represent R11xe2x80x94COO (in which R11 is C1-4 alkyl, C1-4 alkoxy, phenyl, phenyl-C1-4 alkyl, R14xe2x80x94OOCxe2x80x94C1-4 alkyl or R14xe2x80x94OOCxe2x80x94C2-4 alkenyl (in which R14 is hydrogen atom or C1-4 alkyl) 8-9 position is double bond), i.e., the compounds of formula (IC) 
xe2x80x83wherein all symbols are the same meaning as hereinbefore defined.
In formula (I) or (IC), C1-4 alkyl represented by R3, R11, and R14 means methyl, ethyl, propyl, butyl and isomers thereof.
In formula (I), (IA) or (IB), C1-6 alkyl represented by R10, R12 and R13 means methyl, ethyl, propyl, butyl, pentyl, hexyl and isomers thereof.
In formula (I), C1-8 alkyl represented by R3 means methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl and isomers thereof.
In formula (I), C2-4 alkenyl represented by R11 means vinyl, propenyl, butenyl and isomers thereof.
In formula (I), C2-8 alkenyl represented by R3 means vinyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl and isomers thereof.
In formula (I), C2-8 alknyl represented by R3 means ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl and isomers thereof.
In formula (I), or (IC), C1-4 alkoxy represented by R2, R11 and R3 means methoxy, ethoxy, propoxy, butoxy and isomers thereof.
In formula (I), C3-7 cycloalkyl represented by R3 means cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.
In formula (I), a halogen atom represented by R1 and R3 means fluorine, chlorine, bromine and iodine.
In the present invention, it may be easily understood by those skilled in the art, unless otherwise specified, the symbol:
indicates that the substituent attached thereto is in front of the sheet, unless otherwise specified, the symbol:
indicates that the substituent attached thereto is behind the sheet, unless otherwise specified, the symbol:
indicates that the substituent attached thereto is a mixture of in front of and behind the sheet or may be in front of or behind the sheet.
Unless otherwise specified, all isomers are included in the present invention. For example, the alkyl, alkenyl and alkynyl groups include straight-chain and also branched-chain ones. The double bond in alkenyl group include E, Z and EZ mixture ones. Isomers generated by the existence of asymmetric carbon atom(s) e.g. in are included in branched-chain alkyl are included in the present invention.
Preferred compounds of the present invention of the formula (I) are listed in examples, Table 1-14 or prodrug thereof.
Salts
The compounds of formula(I) of the present invention may be converted into a corrresponding non-toxic salt by methods known per se. Non toxic and water-soluble salts are preferable. Suitable salts, for example, are salts of an alkaline metal (potassium, sodium, etc.), salts of an alkaline earth metal (calcium, magnesium, etc.), ammonium salts and salts of pharmaceutically-acceptable organic amines (tetramethylammonium, triethylamine, methylamine, dimethylamine, cyclopentylamine, benzylamine, phenethylamine, piperidine, monoethanolamine, diethanolamine, tris(hydroxymethyl)methylamine, lysine, arginine, N-methyl-D-glucamine, etc.).
Cyclodextrin Clathrates
Cyclodextrin clathrates of xcfx89-cycloalkyl-prostaglandin E2 derivatives of the formula (I) may be prepared by the method described in the specification of GB 1351238, which is herein incorporated by reference, using xcex1-, xcex2- or xcex3-cyclodextrins or a mixture thereof. Converting into their cyclodextrin clathrates serves to increase the stability and solubility in water of the compounds, and is therefore, useful in the use of pharmaceuticals.
1) For compounds of formula (I) of the present invention, those in which R is carboxy, i.e., the compounds of formula (I-1) 
wherein all the symbols are the same meaning hereinbefore defined may be prepared by hydrolysis using enzyme or hydrolysis in an alkaline condition of a compound of formula (IA) 
wherein all the symbols are the same meaning hereinbefore defined.
The hydrolysis using enzyme is known. For example, hydrolysis may be carried out in the mixture of a water-miscible organic solvent (ethanol, dimethylsulfoxide etc.) and water, in the presence or absence of buffer, using an ester cleaving enzyme (esterase, lipase etc.), at a temperature of from 0xc2x0 C. to 50xc2x0 C.
The hydrolysis in an alkaline condition is known. For example, hydrolysis may be carried out in a water-miscible organic solvent (ethanol, tetrahydrofuran, dioxan etc.), using aqueous solution of an alkali (sodium hydroxide, potassium hydroxide, potassium carbonate etc.), at a temperature of from xe2x88x9210 to 90xc2x0 C.
2) For compounds of formula (I) of the present invention, those in which R is hydroxymethyl, i.e., in the compounds of formula (I-2), 
wherein all the symbols are the same meaning as hereinbefore defined;
those in which R1 is oxo, i.e., the compounds of formula (I-2A) 
wherein all the symbols are the same meaning as hereinbefore defined;
may be prepared by subjecting to elimination of the protecting group in an acidic condition of a compound of formula (IIA) 
wherein R20 is hydrogen atom, hydroxy protecting group to elimination in an acidic condition or C1-4 alkoxy, R40 is hydroxy protecting group to remove in an acid condition, the other symbols are the same meaning as herein before defined.
The hydroxy protecting group to elimination in an acidic condition include, for example, t-butyldimethylsiliyl, triphenylmethyl, tetrahydropyran-2-yl etc.
The hydrolysis in an acidic condition is known. For example, hydrolysis may be carried out in a water-miscible organic solvent (tetrahydrofuran, methanol, ethanol, dimethoxyethane, acetonitrile or mixture thereof etc.), using an inorganic acid (hydrochloric acid, phosphoric acid, hydrofluoric acid or hydrogen fluoride-pyridine etc.), or organic acid (acetic acid, p-toluenesulfonic acid, trichloroacetc acid, etc.) at a temperature of from 0 to 50xc2x0 C.
3) For compounds of formula (I) of the present invention, those in which R is hydroxymethyl, i.e., in the compounds of formula (I-2), 
wherein all the symbols are the same meaning as hereinbefore defined;
those in which R1 is methylene, i.e., the compounds of formula (I-2B) 
wherein all the symbols are the same meaning as hereinbefore defined,
may be prepared by subjecting reduction of a compound of formula (IA-4) 
wherein all the symbols are the same meaning as hereinbefore defined.
The reduction is known. For example, reduction may be carried out in an inert organic solvent (tetrahydrofuran (THF), hexane, toluene, etc.), using diisobutylaluminum hydride at a temperature of from xe2x88x9280 to 0xc2x0 C.
4) For compounds of formula (I) of the present invention, those in which R is hydroxymethyl, i.e., in the compounds of formula (I-2), 
wherein all the symbols are the same meaning as hereinbefore defined;
those in which R1 is halogen atom, i.e., the compounds of formula (I-2C) 
wherein R15 is halogen atom, the other symbols are the same meaning as hereinbefore defined;
may be prepared by subjecting reduction of a compound of formula (IA-5) 
wherein all the symbols are the same meaning as hereinbefore defined.
The reduction may be carried out by the same method as hereinbefore described.
5) For prodrug compounds of formula (IA) of the present invention, those in which R2 is hydrogen atom or hydroxy, i.e., the compounds of formula (IA-1) 
wherein R22 is hydrogen atom or hydroxy, the other symbols are the same meaning as hereinbefore defined;
may be prepared by subjecting hydrolysis in an acidic condition of a compound of formula (III) 
wherein R21 is hydrogen atom or hydroxy protecting group to elimination in an acid condition, the other symbols are the same meaning as hereinbefore defined.
The hydrolysis in an acidic condition may be carried out by the same method as hereinbefore described.
6) For prodrug compounds of formula (IA) of the present invention, those in which R2 is C1-4 alkoxy, i.e., the compounds of formula (IA-2) 
wherein R23 is C1-4 alkoxy, the other symbols are the same meaning as hereinbefore defined;
may be prepared by subjecting O-alkylation of the compounds of the formula (IA-1) those in which R22 is hydroxy, i.e., a compound of formula (IA-3) 
wherein all the symbols are the same meaning as hereinbefore defined.
O-alkylation is known. For example, O-alkylation may be carried out in an inert organic solvent (THF, diethyl ether, etc.), using diazoalkane at a temperature of from xe2x88x9230 to 40xc2x0 C. or in an inert organic solvent (acetonitrile, etc.), in the presence of silver oxide, using alkyl iodide at a temperature of from 0 to 40xc2x0 C.
7) The prodrug compounds of formula (IB) of the present invention may be prepared by subjecting amidation of the compounds of the formula (I-1) 
wherein all the symbols are the same meaning as hereinbefore defined;
with the compounds of the formula (IV) 
wherein all the symbols are the same meaning as hereinbefore defined.
Amidation is known. For example, amidation may be carried out in an inert organic solvent (THF, dichloromethane, benzene, acetone, acetonitrile or mixture thereof etc.), in the presence or absence of tertiary amine(dimethylaminopyridine, pyridine, triethylamine, etc.), using condensing agent (1,3-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC), etc.) at a temperature of from 0 to 50xc2x0 C.
8) The prodrug compounds of formula (IC) of the present invention may be prepared by subjecting hydrolysis in an acidic condition of the compounds of the formula (V) 
wherein all the symbols are the same meaning as hereinbefore defined.
The hydrolysis in an acidic condition may be carried out by the same method as hereinbefore described.
The compound of the formula (IIA) may be prepared by according to the reaction of the following Scheme (J).
The compound of the formula (V) may be prepared by according to the reaction of the following Scheme (K).
The compound of the formula (III) may be separated according to the type of R1 and R21 into the following six types of compounds. That is,
1) R1 is oxo, R21 is hydroxy protecting group to elimination in an acidic condition, i.e., the compound of formula (IIIA) 
wherein R24 is hydroxy protecting group to elimination in an acidic condition, the other symbols are the same meaning as hereinbefore defined,
2) R1 is methylene, R21 is hydroxy protecting group to elimination in an acidic condition, i.e., the compound of formula (IIIB) 
wherein all symbols are the same meaning as hereinbefore defined,
3) R1 is halogen atom, R24 is hydroxy protecting group to elimination in an acidic condition, i.e., the compound of formula (IIIC) 
wherein R15 is halogen atom, the other symbols are the same meaning as hereinbefore defined,
4) R1 is oxo, R21 is hydrogen atom, i.e., the compound of formula (IIID) 
wherein all symbols are the same meaning as hereinbefore defined,
5) R1 is methylene, R21 is hydrogen atom, i.e., the compound of formula (IIIE) 
wherein all symbols are the same meaning as hereinbefore defined,
6) R1 is halogen atom, R21 is hydrogen atom, i.e., the compound of formula (IIIF) 
wherein all symbols are the same meaning as hereinbefore defined.
The compound of the formula (IIIB) may be prepared from the compound of the formula (IIIA) according to the reaction of the following Scheme (A).
The compound of the formula (IIIC) may be prepared from the compound of the formula (IIIA) according to the reaction of the following Scheme (B), (C) or (D).
The compound of the formula (IIID) may be prepared from the compound of the formula (IIIA) according to the reaction of the following Scheme (E).
The compound of the formula (IIIE) may be prepared from the compound of the formula (IIID) according to the same reaction of the following Scheme (A).
The compound of the formula (IIIF) may be prepared from the compound of the formula (IIID) according to the same reaction of the following Scheme (B), (C) or (D).
The compound of the formula (IIIA) may be prepared by according to the reaction of the following Scheme (F), (G) or (H).
In the Scheme, the symbols represent meanings as follow, or the same meaning as hereinbefore described.
Ts is p-toluenesulfonyl;
Ac is acetyl;
Ph is phenyl;
AIBN is 2,2xe2x80x2-azobisisobutylonitrile;
DIBAL is diisobutylaluminum hydride;
t-Bu is t-butyl;
n-Bu is normal butyl;
c-Hex is cyclohexyl;
Et is ethyl;
EE is ethoxyethyl;
D-(xe2x88x92)-DIPT is D-(xe2x88x92)-diisopropyl tartarate;
L-(+)-DIPT is L-(+)-diisopropyl tartarate;
Ti(OiPr)4 is titanium (IV) isopropoxide;
TBHP is t-butylhydroperoxide;
Cp2ZrClH is bis(cyclopentadienyl)zirconium chloride hydride. 
Each reaction of hereinbefore described reaction Scheme may be carried out known methods. In the reaction Scheme, The compound of formula (VI), (VIII), (X), (XII), (XIII), (XI) and (XVI) as starting materials are known per se or may be prepared by known methods.
For example, in the compound of formula (VI), (4RS)-5,5-propanooct-1-yn-4-ol is known compound described in the specification of U.S. Pat. No. 4,132,738.
In the compound of formula (VII), (5Z)-7-((3R)-3-t-butyldimethylsilyloxy-5-oxocyclopent-1-ene)hept-5-enoic acid methylester and in the compound of formula (X), (4R)-2-(diethylaminomethyl)-4-t-butyldimethylsilyloxy-2-cyclopenten-1-one is known compound described in the literature of J. Org. Chem., 53, 5590-5592 (1988).
In the compound of formula (XII), (4R)-4-t-butyldimethylsilyloxy-2-cyclopenten-1-one and in the compound of formula (XIII), 7-iodohept-5-ynoic acid methylester is known compound described in the literature of J. Am. Chem. Soc., 110, No. 14, 4718-4726 (1988).
The compound of formula (XI) is known compound described in the literature of J. Am. Chem. Soc., 97, 4745-4746 (1975).
The starting materials and reagents in the present invention are known per se or may be prepared by known methods.
In each reaction in the present specification, obtained products may be purified by conventional techniques. For example, purification may be carried out by distillation at atmospheric or reduced pressure, by high performance liquid chromatography, by thin layer chromatography or by column chromatography using silica gel or magnesium silicate, by washing or by recrystallization. Purification may be carried out after each reaction, or after a series of reactions.
Pharmacological Activities
The compounds of the present invention of the formula (I) bind and act on EP2 receptor which is a subtype of of PGE2 receptor.
For example, in standard laboratory test, the effects of the compounds of the present invention were confirmed by binding assay using expression cell of prostanoide receptor subtype.
Binding Assay Using Expression Cell of Prostanoide Receptor Subtype
The preparation of membrane fraction was carried out according to the method of Sugimoto et. al. [J. Biol. Chem. 267, 6463-6466 (1992)], using expression CHO cell of the prostanoide receptor subtype (mouse EP1, EP2, EP3xcex1, EP4).
The standard assay mixture contained membrane fraction (0.5 mg/ml), and [3H]-PGE2 in a final volume of 200 xcexcl was incubated for 1 hour at room temperature. The reaction was terminated by the addition of 3 ml of ice-cold buffer. The mixture was rapidly filtered through a GF/B glass filter. The radioactivity associated with the filter was measured by liquid scintillation counting.
Kd and Bmax values were determined from Scatchard plots [Ann. N.Y. Acad. Sci., 51, 660 (1949)]. Non-specific binding was calculated as the bound in the presence of an excess (2.5 xcexcM) of unlabeled PGE2. In the experiment for competition of specific 3H-PGE2 binding by the compounds of the present inventions 2.5 nM of 3H-PGE2 and various concentrations of compounds of the present invention were added. The following buffer was used in all reactions.
Buffer: 10 mM potassium phosphate (pH 6.0), 1 mM EDTA, 10 mM MgCl2, 0.1 M NaCl
In the example compounds, all of values were shown the more polar compounds.
The dissociation constant (Ki) of each compound was calculated by the following equation.
xe2x80x83Ki=IC50/(1+([C]/Kd))
The results were shown in Table 15.
Toxicity
The toxicity of the compounds of the present invention are very low and therefore, it is confirmed that these compounds are safe for pharmaceutical use.
The compounds of the present invention of the formula (I) bind strongly and act on PGE2 receptor, especially on EP2 subtype receptor and therefore are useful for prevention and/or treatment of immunologic diseases (autoimmune diseases, organ transplantation, etc.), asthma, abnormal bone formation, neuronal cell death, liver damage, abortion, premature birth or retina neuropathy of glaucoma etc.
Among the compounds of the present invention of the formula (I), compounds which bind weakly on to receptor subtypes except for EP2 receptors and another arachidonic acid metabolism receptors (thromboxane receptor, PGI2 receptor, etc.) do not express other effects and therefore it is thought that such compounds will be a medical agent which have less side-effects.
For the purpose above described, the compounds of the formula (I), (IA), (IB) and (IC), prodrug thereof, non-toxic salts thereof and cyclodextrin clathrate thereof may be normally administered systematically or partially, usually by oral or parenteral administration. To convert prodrug, they have merit of non-stimulant, good-absorbability, good-solubility, etc.
The doses to be administered are determined depending upon age, body weight, symptom, the desired therapeutic effect, the route of administration, and the duration of the treatment etc. In the human adult, the doses per person per dose are generally between 1 xcexcg and 100 mg, by oral administration, up to several times per day, and between 0.1 xcexcg and 10 mg, by parenteral administration (preferred into vein) up to several times per day, or continuous administration between 1 and 24 hrs. per day into vein.
As mentioned above, the doses to be used depend upon various conditions. Therefore, there are cases in which doses lower than or greater than the ranges specified above may be used.
When administering the compounds of the present invention, it is used as solid compositions, liquid compositions or other compositions for oral administration, as injections, liniments or suppositories etc. for parenteral administration.
Solid compositions for oral administration include compressed tablets, pills, capsules, dispersible powders, and granules.
Capsules contain hard capsules and soft capsules.
In such compositions, one or more of the active compound(s) is or are, admixed with at least one inert diluent such as lactose, mannitol, mannit, glucose, hydroxypropyl cellulose, microcrystalline cellulose, starch, polyvinylpyrrolidone, magnesium metasilicate aluminate. The compositions may also comprise, as is normal practice, additional substances other than inert diluents: e.g. lubricating agents such as magnesium stearate, disintegrating agents such as cellulose calcium glycolate, and assisting agents for dissolving such as glutamic acid, asparaginic acid. The tablets or pills may, if desired, be coated with film of gastric or enteric material such as sugar, gelatin, hydroxypropyl cellulose or hydroxypropyl cellulose phthalate etc., or be coated with two or more films. And further, coating may include containment within capsules of absorbable materials such as gelatin.
Liquid compositions for oral administration include pharmaceutically-acceptable emulsions, solutions, syrups and elixirs etc. In such liquid compositions, one or more of the active compound(s) is or are comprised in inert diluent(s) commonly used in the art (for example, purified water, ethanol etc.). Besides inert diluents, such compositions may also comprise adjuvants such as wetting agents, suspending agents, sweetening agents, flavouring agents, perfuming agents and preserving agents.
Other compositions for oral administration include spray compositions which may be prepared by known methods and which comprise one or more of the active compound(s). Spray compositions may comprise additional substances other than inert diluents: e.g. stabilizing agents such as sodium hydrogen sulfate, stabilizing agents to give isotonicity, isotonic buffer such as sodium chloride, sodium citrate, citric acid. For preparation of such spray compositions, for example, the method described in the U.S. Pat. No. 2,868,691 or U.S. Pat. No. 3,095,355 may be used.
Injections for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Aqueous solutions or suspensions include distilled water for injection and physiological salt solution. Non-aqueous solutions or suspensions include propylene glycol, polyethylene glycol, plant oil such as olive oil, alcohol such as ethanol, POLYSOLBATE80 (registered trade mark) etc. Such compositions may comprise additional diluents: e.g. preserving agents, wetting agents, emulsifying agents, dispersing agents, stabilizing agent, assisting agents such as assisting agents for dissolving (for example, glutamic acid, asparaginic acid). They may be sterilized for example, by filtration through a bacteria-retaining filter, by incorporation of sterilizing agents in the compositions or by irradiation. They may also be manufactured in the form of sterile solid compositions and which can be dissolved in sterile water or some other sterile diluents for injection immediately before used.
Other compositions for parenteral administration include liquids for external use, and endemic liniments, ointment, suppositories and pessaries which comprise one or more of the active compound(s) and may be prepared by know methods.